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By Authority Of 


Legally Binding Document 

By the Authority Vested By Part 5 of the United States Code § 552(a) and 
Part 1 of the Code of Regulations § 51 the attached document has been duly 
INCORPORATED BY REFERENCE and shall be considered legally 
binding upon all citizens and residents of the United States of America. 
HEED THIS NOTICE : Criminal penalties may apply for noncompliance. 

Document Name: APHA Method 9215: Standard Methods for the 

Examination of Water and Wastewater 

CFR Section(s) : 40 CFR 141121 

Standards Body: American Public Health Association 


For the 

Examination of 
Water and 

: mm 


Prepared and published jointly by: 

American Public Health Association 

American Water Works Association 

Water Environment Federation 

joint Editorial Board 

Arnold E. Greenberg, APHA, Chairman 

Lenore S. Qesceri, WEF 

Andrew D. Eaton, AWWA 

Managing Editor 
Mary Ann H. Franson 

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Copyright ° 1971 by 

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Copyright® 1976 by 
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Water Pollution Control Federation 

Copyright © 1 981 by 
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Copyright & 1985 by 
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Water Pollution Control Federation 

Copyright © 1989 by 
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Water Pollution Control Federation 

Copyright ° 1992 by 

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Water Environment Federation 

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The Library of Congress has catalogued this work as follows: 
American Public Health Association. 

Standard methods for the examination of water and wastewater. 
ISBN 0-87553-207-1 

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9215 A. Introduction 

1. Applications 

The heterotrophic plate count (HPC), formerly known as the 
standard plate count, is a procedure for estimating the number 
of live heterotrophic bacteria in water and measuring changes 
during water treatment and distribution or in swimming pools. 
Colonies may arise from pairs, chains, clusters, or single cells, 
all of which are included in the term '^colony-forming units" 
(CFU). The final count also depends on interaction among the 
developing colonies; choose that combination of procedure and 
medium that produces the greatest number of colonies within 
the designated incubation time. Three different methods and four 
different media are described. 

2. Selection of Method 

guished readily from particles and bubbles. Colonies can be 
transferred quickly and compared easily to published descrip- 
tions. However, this method is limited by the small volume of 
sample or diluted sample that can be absorbed by the agar: 0.1 
to 0.5 mL, depending on the degree to which the prepoured 
plates have been dried. To use this procedure, maintain a supply 
of suitable predried, absorbent agar plates. 

The membrane filter method (9215D) permits testing large 
volumes of low-turbidity water and is the method of choice for 
low-count waters (< 1 to 10 CFU/mL). This method produces 
no heat shock but adds the expense of the membrane filter. 
Further disadvantages include the smaller display area, the need 
to detect colonies by reflected light against a white background 
if colored filters or contrast stains are not used, and possible 
variations in membrane filter quality (see Section 9020B.3g). 

The pour plate method (9215B) is simple to perform and can 
accommodate volumes of sample or diluted sample ranging from 
0.1 to 2.0 mL. The colonies produced are relatively small and 
compact, showing less tendency to encroach on each other than 
those produced by surface growth. On the other hand, sub- 
merged colonies often are slower growing, are difficult to trans- 
fer, and are not described in published studies. A thermostati- 
cally controlled water bath is essential for tempering the agar, 
but even so, significant heat shock from the transient exposure 
of the sample to 45 to 46°C agar may occur. 

The spread plate method (9215C) causes no heat shock and 
all colonies are on the agar surface where they can be distin- 

: Approved by Standard Methods Committee, 1988. 

3. Work Area 

Provide a level table or bench top with ample area in a clean, 
draft-free, well-lighted room or within a horizontal-flow laminar 
hood. Use table and bench tops having nonporous surfaces and 
disinfect before any analysis is made. 

4. Samples 

Collect water as directed in Section 9060A. Initiate analysis 
as soon as possible after collection to minimize changes in bac- 
terial population. The recommended maximum elapsed time be- 
tween collection and examination of samples is 8 h (maximum 
transit time 6 h, maximum processing time 2 h). When analysis 
cannot begin within 8 h, maintain sample at a temperature below 

HETEROTROPHIC PLATE COUNT (9215)/lntroduction 


4°C but do not freeze. Do not let maximum elapsed time between 
collection and analysis exceed 24 h. 

Hold or transport bottled water samples obtained from retail 
outlets unrefrigerated under the same ambient conditions as those 
found in the retail store until tested. Examine freshly bottled 
samples (less than 48 h old) within 6 h of collection if unre- 
frigerated and within 24 h if refrigerated. 

5. Sample Preparation 

Mark each plate with sample number, dilution, date, and any 
other necessary information before examination. Prepare at least 
duplicate plates for each volume of sample or dilution examined. 
For the pour or spread plate methods use glass (65 cm 2 ) or 
disposable plastic (57 cm 2 ) petri dishes. 

Thoroughly mix all samples or dilutions by rapidly making 
about 25 complete up-and-down (or back-and-forth) movements. 
Optionally, use a mechanical shaker to shake samples or dilutions 
for \5 s. 

6. Media 

Compare new lots of media with current lot in use according 
to Section 9020B.3/z. 

a. Plate count agar (tryptone glucose yeast agar): Use for pour 
and spread plate methods. This high-nutrient agar, widely used 
in the past, gives lower counts than R2A or NWRI agar. It is 
included for laboratories wishing to make media comparisons or 
to extend the continuity of old data. 

Tryptone 5.0 g 

Yeast extract 2.5 g 

Glucose 1.0 g 

Agar 15.0 g 

Reagent-grade water 1 L 

pH should be 7.0 ± 0.2 after autoclaving at 121°C for 15 min. 

b. m-HPC agarr\ Use this high-nutrient medium only for the 
membrane filter method. 

Peptone 20.0 g 

Gelatin 25.0 g 

Glycerol 10.0 mL 

Agar 15.0 g 

Reagent-grade water 1 L 

Mix all ingredients except glycerol. Adjust pH to 7.1, if nec- 
essary, with \N NaOH, heat to dissolve, add glycerol, and au- 
toclave at 121°C for 5 min. 

c. R2A agar; Use for pour, spread plate, and membrane filter 
methods. This low-nutrient agar gives higher counts than high- 
nutrient formulations. 

Yeast extract 0.5 g 

Proteose peptone No. 3 or polypeptone 0.5 g 

Casamino acids 0.5 g 

Glucose 0.5 g 

Soluble starch 0.5 g 

Dipotassium hydrogen phosphate, K 2 HP0 4 0.3 g 

Formerly called m-SPC a car. 

Magnesium sulfate heptahydrate, MgS0 4 -7H 2 0.05 g 

Sodium pyruvate 0.3 g 

Agar 15.0 g 

Reagent-grade water 1 L 

Adjust pH to 7.2 with solid K 2 HP0 4 or KH 2 P0 4 before adding 
agar. Heat to dissolve agar and sterilize at 121°C for 15 min. 

d. NWRI agar (HPCA): Use for pour, spread plate, and mem- 
brane filter methods. This medium is likely to produce higher 
colony counts than the three media described above. It may not 
be available in dehydrated form and may require preparation 
from the basic ingredients. 

Peptone 3.0 g 

Soluble casein 0.5 g 

K 2 HP0 4 0.2 g 

MgSO, 0.05 g 

FeCI, 0.001 g 

Agar 15.0 g 

Reagent-grade water 1 L 

Adjust pH to 7.2 before autoclaving for 15 min at 121°C. 

7. Incubation 

U.S. EPA revised regulations will describe the pour plate 
method. In testing to meet these regulations, incubate at 35°C 
for 48 h. Otherwise, select from among recommended times and 
temperatures for monitoring changes in water quality. The high- 
est counts typically will be obtained from 5- to 7-d incubation at 
a temperature of 20 to 28°C. If unable to provide incubators 
controlled at 20 to 28°C, use a dust-free, room-temperature cab- 
inet. Include time and temperature in reporting results. 

During incubation maintain humidity within the incubator so 
that plates will have no moisture weight loss greater than 15%. 
This is especially important if prolonged incubation is used. A 
pan of water placed at the bottom of the incubator may be 
sufficient but note that to prevent rusting or oxidation the inside 
walls and shelving should be of high-grade stainless steel or an- 
odized aluminum. For long incubation in nonhumidified incu- 
bators, seal plates in plastic bags. 

8. Counting and Recording 

a. Pour and spread plates: Count all colonies on selected plates 
promptly after incubation. If counting must be delayed tempo- 
rarily, store plates at 5 to 10°C for no more than 24 h, but avoid 
this as routine practice. Record results of sterility controls on 
the report for each lot of samples. 

Use an approved counting aid, such as the Quebec colony 
counter, for manual counting. If such equipment is not available, 
count with any other counter provided that it gives equivalent 
magnification and illumination. Automatic plate counting in- 
struments are available. These generally use a television scanner 
coupled to a magnifying lens and an electronics package. Their 
use is acceptable if evaluation in parallel with manual counting 
gives comparable results. 

In preparing plates, plant sample volumes that will give from 
30 to 300 colonies/plate. The aim is to have at least one dilution 
giving colony counts between these limits, except as provided 



Ordinarily, do not plant more than 2.0 mL of sample; there- 
fore, when the total number of colonies developing from 2.0 mL 
is less than 30, disregard the rule above and record result ob- 
served. With this exception, consider only plates having 30 to 
300 colonies in determining the plate count. Compute bacterial 
count per milliliter by multiplying average number of colonies 
per plate by the reciprocal of the dilution used. Report counts 
as CFU per milliliter. 

If there is no plate with 30 to 300 colonies, and one or more 
plates have more than 300 colonies, use the plate(s) having a 
count nearest 300 colonies. Compute the count by multiplying 
average count per plate by the reciprocal of the dilution used 
and report as estimated CFU per milliliter. 

If plates from all dilutions of any sample have no colonies, 
report the count as less than one (< 1) times the reciprocal of 
the corresponding lowest dilution. For example, if no colonies 
develop on the 1:100 dilution, report the count as less than 100 
(< 100) estimated CFU/mL. 

If the number of colonies per plate far exceeds 300, do not 
report result as "too numerous to count 1 ' (TNTC). If there are 
fewer than 10 colonies/cm 2 , count colonies in 13 squares (of the 
colony counter) having representative colony distribution. If pos- 
sible, select seven consecutive squares horizontally across the 
plate and six consecutive squares vertically, being careful not to 
count a square more than once. Multiply sum of the number of 
colonies in 13 representative square centimeters by 5 to compute 
estimated colonies per plate when the plate area is 65 cm 2 . When 
there are more than 10 colonies /cm 2 , count four representative 
squares, take average count per square centimeter, and multiply 
by the appropriate factor to estimate colonies per plate. The 
factor is 57 for disposable plastic plates and 65 for glass plates. 
When bacterial counts on crowded plates are greater than 100 
colonies/cm 2 , report result as greater than (>) 6500 times the 
reciprocal of the highest dilution plated for glass plates or greater 
than (>) 5700 times the reciprocal for plastic plates. Report as 
estimated colony-forming units per milliliter. 

If spreading colonies (spreaders) are encountered on the plate(s) 
selected, count colonies on representative portions only when 
colonies are well distributed in spreader-free areas and the area 
covered by the spreader(s) does not exceed one-half the plate 

When spreading colonies must be counted, count each of the 
following types as one: a chain of colonies that appears to be 
caused by disintegration of a bacterial clump as agar and sample 
were mixed; a spreader that develops as a film of growth between 
the agar and bottom of petri dish; and a colony that forms in a 
film of water at the edge or over the agar surface. The last two 
types largely develop because of an accumulation of moisture at 
the point from which the spreader originates. They frequently 
cover more than half the plate and interfere with obtaining a 
reliable plate count. 

Count as individual colonies similar-appearing colonies grow- 
ing in close proximity but not touching, provided that the distance 
between them is at least equal to the diameter of the smallest 
colony. Count impinging colonies that differ in appearance, such 
as morphology or color, as individual colonies. 

If plates have excessive spreader growth, report as "spreaders" 
(Spr). When plates are uncountable because of missed dilution, 
accidental dropping, and contamination, or the control plates 
indicate that the medium or other material or labware was con- 
taminated, report as "laboratory accident' 1 (LA). 

b. Membrane filter method: Count colonies on membrane fil- 
ters using a stereoscopic microscope at 10 to 15 x magnification. 
Preferably place petri dish on microscope stage slanted at 45° 
and adjust light source vertical to the colonies. Optimal colony 
density per filter is 20 to 200. If colonies are small and there is 
no crowding, a higher limit is acceptable. 

Count all colonies on the membrane when there are 1 to 2, 
or fewer, colonies per square. For 3 to 10 colonies per square 
count 10 squares and obtain average count per square. For 10 
to 20 colonies per square count 5 squares and obtain average 
count per square. Multiply average count per square by 100 times 
the reciprocal of the dilution to give colonies per milliliter. If 
there are more than 20 colonies per square, record count as 
> 2000 times the reciprocal of the dilution. Report averaged 
counts as estimated colony-forming units. Make estimated counts 
only when there are discrete, separated colonies without spread- 

9. Computing and Reporting Counts 

The term "colony-forming units' 1 (CFU) is descriptive of the 
methods used; therefore, report all counts as colony-forming 
units. Include in the report the method used, the incubation 
temperature and time, and the medium. For example: CFU/mL, 
pour plate method, 35°C/48 h, plate count agar or 28°/5 d, R2A 
agar; or CFU/mL, spread plate method, 2077 d, NWRI agar. 

To compute the heterotrophic plate count, CFU/mL, multiply 
total number of colonies or average number (if duplicate plates 
of the same dilution) per plate by the reciprocal of the dilution 
used. Record dilutions used and number of colonies on each 
plate counted or estimated. 

When colonies on duplicate plates and/or consecutive dilutions 
are counted and results are averaged before being recorded, 
round off counts to two significant figures only when converting 
to colony-forming units. 

Avoid creating fictitious precision and accuracy when com- 
puting colony-forming units by recording only the first two left- 
hand digits. Raise the second digit to the next higher number 
when the third digit from the left is 5, 6, 7, 8, or 9; use zeros 
for each successive digit toward the right from the second digit. 
For example, report a count of 142 as 140 and a count of 155 as 
160, but report a count of 35 as 35. 

10. Personal Errors 

Avoid inaccuracies in counting due to carelessness, damaged 
or dirty optics that impair vision, or failure to recognize colonies. 
Laboratory workers who cannot duplicate their own counts on 
the same plate within 5% and the counts of other analysts within 
10% should discover the cause and correct such disagreements. 


9215 B. Pour Plate Method 


1 . Samples and Sample Preparation 
See 9215A.4 and 9215A.5. 

2. Sample Dilution 

Prepare water used for dilution blanks as directed in Section 

a. Selecting dilutions: Select the dilution(s) so that the total 
number of colonies on a plate will be between 30 and 300 (Figure 
9215:1). For example, where a heterotrophic plate count as high 
as 3000 is suspected, prepare plates with 10~ 2 dilution. 

For most potable water samples, plates suitable for counting 
will be obtained by plating 1 mL and 0.1 mL undiluted sample 
and 1 mL of the 10 2 dilution. 

b. Measuring sample portions: Use a sterile pipet for initial 
and subsequent transfers from each container. If pipet becomes 
contaminated before transfers are completed, replace with a ster- 
ile pipet. Use a separate sterile pipet for transfers from each 
different dilution. Do not prepare dilutions and pour plates in 
direct sunlight. Use caution when removing sterile pipets from 
the container; to avoid contamination, do not drag pipet tip 
across exposed ends of pipets in the pipet container or across 
lips and necks of dilution bottles. When removing sample, do 
not insert pipets more than 2.5 cm below the surface of sample 
or dilution. 

c. Measuring dilutions: When discharging sample portions, hold 
pipet at an angle of about 45° with tip touching bottom of petri 
dish or inside neck of dilution bottle. Lift cover of petri dish just 
high enough to insert pipet. Allow 2 to 4 s for liquid to drain 
from 1-mL graduation mark to tip of pipet. If pipet is not a blow- 
out type, touch tip of pipet once against a dry spot on petri dish 
bottom. Less preferably, use a cotton-plugged blow-out-type pi- 

pet and gently blow out remaining volume of sample dilution. 
When 0.1-mL quantities are measured, let diluted sample drain 
from chosen reference graduation until 0.1 mL has been deliv- 
ered. Remove pipet without retouching it to dish. Pipet 1 mL, 
0.1 mL, or other suitable volume into sterile petri dish before 
adding melted culture medium. Use decimal dilutions in pre- 
paring sample volumes of less than 0.1 mL; in examining sewage 
or turbid water, do not measure a 0.1-mL inoculum of original 
sample, but prepare an appropriate dilution. Prepare at least two 
replicate plates for each sample dilution used. After depositing 
test portions for each series of plates, pour culture medium and 
mix carefully. Do not let more than 20 min elapse between start- 
ing pipetting and pouring plates. 

3. Plating 

a. Melting medium: Melt sterile solid agar medium in boiling 
water or by exposure to flowing steam in a partially closed con- 
tainer, but avoid prolonged exposure to unnecessarily high tem- 
peratures during and after melting. Do not resterilize plating 
medium. If the medium is melted in two or more batches, use 
all of each batch in order of melting, provided that the contents 
remain fully melted. Discard melted agar that contains precipi- 

Maintain melted medium in a water bath between 44 and 46°C 
until used. In a separate container place a thermometer in water 
or medium that has been exposed to the same heating and cooling 
as the plating medium. Do not depend on the sense of touch to 
indicate proper medium temperature when pouring agar. 

Use plate count agar, R2A agar, or NWRI agar as specified 
in Section 9215A.6. Before using a new lot of medium test its 

Delivery volume 

Culture dishes 

Actual volume oi' 
sample in dish 

1 mL 0.1 mL 10" 2 mL 

Figure 9215:1. Preparation of dilutions. 

10 ; ' mL 



b. Pouring plates: Limit the number of samples to be plated 
in any one series so that no more than 20 min (preferably 10 
min) elapse between dilution of the first sample and pouring of 
the last plate in the series. Pour at least 10 to 12 mL liquefied 
medium maintained at 44 to 46°C into each dish by gently lifting 
cover just high enough to pour. Carefully avoid spilling medium 
on outside of container or on inside of dish lid when pouring. 
As each plate is poured mix melted medium thoroughly with test 
portions in petri dish, taking care not to splash mixture over the 
edge, by rotating the dish first in one direction and then in the 
opposite direction, or by rotating and tilting. Let plates solidify 
(within 10 min) on a level surface. After medium solidifies, invert 
plates and place in incubator. 

c. Sterility controls: Check sterility of medium and dilution 
water blanks by pouring control plates for each series of samples. 
Prepare additional controls to determine contamination of plates, 
pipets, and room air. 

4. Incubation 

See Section 9215A.7. 

5. Counting, Recording, Computing, and Reporting 
See Sections 9215A.8 and 9215A.9. 

6. Bibliography 

Breed, R.S. & W.D. Dotterer. 1916. The number of colonies allow- 
able on satisfactory agar plates. Tech. Bull. 53, New York Agri- 
cultural Experiment Sta. 

Butterfield, C.T. 1933. The selection of a dilution water for bacte- 
riological examinations. J. BacterioL 23:355; Pub. Health Rep. 48:681. 

Archambault, J., J. Curot & M.H. McCrady. 1937. The need of 
uniformity of conditions for counting plates (with suggestions for a 
standard colony counter). Amer. J. Pub. Health 27:809. 

Richards, O.W. & P.C. Heijn. 1945. An improved dark- field Quebec 
colony counter. J. Milk Tech not. 8:253. 

Berry, J.M., D.A. McNeill & L.D. Witter. 1969. Effect of delays 
in pour plating on bacterial counts. J. Dairy Sci. 52:1456. 

Geldreich, E.E., H.D. Nash, D.J. Reasoner& R.H. Taylor. 1972. 
The necessity of controlling bacterial populations in potable waters: 
Community water supply. J. Amer. Water Works Assoc. 64:596. 

Geldreich, E.E. 1973. fs the total count necessary? Proc. 1st Annu. 
Water Quality Teehnol. Conf. , American Water Works Assoc, Pa- 
per No. VIM. 

Ginsburg, W. 1973. Improved total count techniques. Proc. 1st Annu. 
Water Quality Teehnol. Conf., American Water Works Assoc, Pa- 
per No. VTIL 

Dutka, B.J., A.S.Y. Chau & J. Coburn. 1974. Relationship of het- 
erotrophic bacterial indicators of water pollution and fecal sterols. 
Water Res. 8:1047. 

Klein, D.A. & S. Wu. 1974. Stress: a factor to be considered in het- 
erotrophic microorganism enumeration from aquatic environments. 
Appl. Microbiol. 37:429. 

Geldreich, E.E. , H.D. Nash, D.J. Reasoner & R.H. Taylor. 1975. 
The necessity for controlling bacterial populations in potable waters: 
Bottled water and emergency water supplies. ,/. Amer. Water Works 
Assoc. 67:117. 

Bell, C.R., M.A. Holder-Franklin & M. Franklin. 1980. Heter- 
otrophic bacteria in two Canadian rivers. — I. Seasonal variation in 
the predominant bacterial populations. Water Res. 14:449. 

Reasoner, D.J. & E.E. Geldreich. 1981. fn flue nee of medium, meth- 
ods and incubation time and temperature on the bacterial count of 
potable water. 8 1st Annu. Meeting, American Soc. Microbiology, 
Dallas, Tex., Paper No. N27. 

Means, E.G., L. Hanami, G.F. Ridgway & B.H. Olson. 1981. Eval- 
uating mediums and plating techniques for enumerating bacteria in 
water distribution systems. J. Amer. Water Works Assoc. 73:585. 

American Public Health Association. 1985. Standard Methods for 
the Examination of Dairy Products, 15th ed. American Public Health 
Assoc, Washington, D,C. 

Reasoner, D.J. & E.E. Geldreich. 1985. A new medium for the 
enumeration and subculture of bacteria from potable water. Appl. 
Environ. Microbiol. 49:1. 

9215 C. Spread Plate Method 

1 . Laboratory Apparatus 

a. Glass rods: Bend 4-mm-diam fire-polished glass rods, 200 
mm in length, 45° about 40 mm from one end. Sterilize before 

b. Pipet, glass, 1.1 mL, with tempered, rounded tip. Do not 
use disposable plastic pipets. 

c. Turntable (optional).* 

d. Incubator or drying oven, set at 42°C, or laminar-flow hood. 

2. Media 

3. Preparation of Plates 

Pour 15 mL of the desired medium into sterile 100 x 15 or 
90 x 15 petri dishes; let agar solidify. Predry plates inverted so 
that there is a 2- to 3-g water loss overnight with lids on. See 
Figure 9215:2, Table 9215:1, or Figure 9215:3. Use predried plates 
immediately after drying. For predrying and using plates the 
same day, pour 25 mL agar into petri dish and dry in a laminar- 
flow hood at room temperature (24 to 26°C) with the lid off to 
obtain the desired 2- to 3-g weight loss. See Figure 9215:3. 

See 92 15 A. 6a, c, and d. If R2A agar is used best results are 
obtained at 28°C with 7 d incubation; if NWRI is used, incubate 
at 20°C for 7 d. 

* Fisher Scientific, hand operated, No. 08-758 or Lab- Line motor driven. No. 1580, 
or equivalent. 

4. Procedure 

Prepare sample dilutions as directed in 921 5B. 2. 

a. Glass rod: Pipet 0.1 or 0.5 mL sample onto surface of 
predried agar plate. Using a sterile bent glass rod, distribute 
inoculum over surface of the medium by rotating the dish by 

HETEROTROPHIC PLATE COUNT (9215)/Spread Plate Method 



"•''42* C 

Figure 9215:2. Drying weight loss of 15-mL agar plates stored separately, inverted with lids on. Source: 
Unpublished data. Water Purification Lab., Chicago Dep. Water. 

O Near screen 

S Near screen. 1 2 lids oft 

a Near ouiside edge fids off 

O Near outside edge i 2 lids off 

A Random placement lids off 

Figure 9215:3. Weight loss of 25-mL agar plates (100 X 15 mm) dried separately in a laminar-flow 
hood at room temperature (24 to 26°C), relative humidity (30 to 33%), and air velocity 

0.6 m/s. Source: Unpublished data. Alberta Environmental Centre, Vengreville, Alta. 



Ta b l n 9215:1. Effect o i ■' Te m p e r atu r f o f D r y inc; on W e i o. ht Loss 
of 15-mL Agar Plates Six) red Separately* 

rime for 

Plates to Lose 

1 to 4 g 

of Water 


for 5 Plates) 


Plates Inverted 

Plates I 



with Lids On 

with Lids 


I g 

2 £ 

3 g 

4 g 


9 a 

3g 4g 








10.5 14.0 








5.3 7.0 








1.9 2.7 








— — 

* Referenced in Canada Centre for Inland Waters Manual. Burl inelon. Out. 

hand or on a turntable. Let inoculum be absorbed completely 
into the medium before incubating. 

b. Piper : Pipet desired sample volume (0.1, 0.5 mL) onto the 
surface of the predried agar plate while dish is being rotated on 
a turntable. Slowly release sample from pipet while making one 
to-and-fro motion, starting at center of the plate and stopping 
0.5 cm from the plate edge before returning to the center. Lightly 
touch the pipet to the plate surface. Let inoculum be absorbed 
completely by the medium before incubating. 

5. Incubation 
See 9215A.7. 

6. Counting, Recording, Computing, and Reporting 
See 9215A.8 and 9215A.9. 

7. Bibliography 

Buck, J.D. & R.C. Clever don. 1960. The spread plate as a method 
for the enumeration of marine bacteria. Umnol. Occanogr. 5:78. 

Clark, D.S. 1967. Comparison of pour and surface plate methods for 
determination of bacterial counts. Can. J. Microbiol. 13:1409. 

Van Soestbergan, A. A. & C.H. Lee. 1969. Pour plates or streak 
plates. Appi. Microbiol. 18:1092. 

Clark, D.S. 1971. Studies on the surface plate method of counting 
bacteria. Can. J. Microbiol J 7:943. 

Gilchrist, J.E., J.E. Campbell, C.B. Donnelly, J.T. Peeler & 
J.M. Delaney. 1973. Spiral plate method for bacterial determi- 
nation. Appi. Microbiol. 25:244. 

Ptak, D.M. & W. Ginsburg. 1976. Pour plate vs. streak plate method. 
Proc. 4th Annu. Water Quality Teehnol. Conf., American Water 
Works Assoc, Paper No. 2B-5. 

Dutka, B.J., ed. 1978. Methods for Microbiological Analysis of Waters, 
Wastewaters and Sediments. Inland Waters Directorate, Scientific 
Operation Div., Canada Centre for Inland Waters, Burlington, On t. 

Kaper, J.B., A.L. Mills & R.R. Colwell. 1978. Evaluation of the 
accuracy and precision of enumerating aerobic heterotrophs in water 
samples by the spread method. Appi. Environ. Microbiol. 35:756. 

Young, M. 1979. A modified spread plate technique for the determi- 
nation of concentrations of viable heterotrophic bacteria. STP 673:41- 
51, American Soc. Testing & Materials. Philadelphia, Pa. 

Geldreich, E.E. 1981. Current status of microbiological water quality 
criteria. ASM News 47:23. 

Taylor, R.H., M..L Allen & E.E. Geldreich. 1981. Standard plate 
count: A comparison of pour plate and spread plate methods. Proc. 
9th Annu. Water Quality Teehnol. Conf., American Water Works 

9215 D. Membrane Filter Method 

1 . Laboratory Apparatus 
See Section 9222B.1. 

2. Media 

See 9215A.6. Use m-HPC agar, or alternatively R2A or NWRI 

3. Preparation of Plates 

Dispense 5-mL portions of sterile medium into 50- x 9-mm 
petri dishes. Let solidify at room temperature. Prepared plates 
may be stored inverted in a plastic bag or tight container in a 
refrigerator, preferably for no longer than 1 week. 

5. Procedure 

Filter appropriate volume through a sterile 47-mm, 0.45-fim, 
gridded membrane filter, under partial vacuum. Rinse funnel 
with three 20- to 30-mL portions of sterile dilution water. Place 
filter on agar in petri dish. 

6. Incubation 

Place dishes in close fitting box or plastic bag containing mois- 
tened paper towels. Incubate at 35 ± 0.5°C for 48 h if using m- 
HPC agar, or longer if using R2A medium, or at 20°C for 7d if 
using NWRI agar. Duplicate plates may be incubated for other 
time and temperature conditions as desired. 

4. Sample Size 

The volume to be filtered will vary with the sample. Select a 
maximum sample size to give 20 to 200 CFU per filter. 

7. Counting, Recording, Computing, and Reporting 

See 9215A.8 and 9215A.9. Report as CFU/mL, membrane 
filter method, time, medium. 

DIRECT TOTAL COUNT (9216)/Epifluorescence Method 


8. Bibliography 

Clark, H.F., E.E. Geldreich, H.L. Jeter Sc P.W. Kabler. 1951. 
The membrane filler in sanitary bacteriology. Pub. Health Rep. 

Stopert, E.M., W.T. Sokoski & J.T. Northam. 1962. The factor of 
temperature in the better recovery of bacteria from water by nitra- 
tion. Can. ./. Microbiol. 8:809. 

Taylor, R.H. & E.E. Geldreich. 1979. A new membrane filter pro- 
cedure for bacterial counts in potable water and swimming pool 
samples. J. A frier. Water Works Assoc. 71:402. 

Clark, J. A. 1980. The influence of increasing numbers of non-indicator 
organisms upon the detection of indicator organisms by the mem- 
brane filter and presence-absence tests. Can. J. Microbiol. 20:827. 

Dutka, B.J., ed. 1981 . Membrane Filtration, Applications, Techniques, 
and Problems. Marcel Dekker, Inc., New York, N.Y. and Basel. 

Hoadley, A.W. 1981. Effect of injury on the recovery of bacteria on 
membrane filters. In B. J. Dutka, ed. Membrane Filtration, Ap- 
plications, Techniques, and Problems, pp. 413-450. Marcel Dekker, 
Inc., New York. N.Y. and Basel, Switzerland. 

d. Blender or vortex mixer. 

: Bausch & Lomb No. 3 1-16- .13. 
■ K. Zeiss or equivalent. 

± Nuclepore Corp. or equivalent. 

§ Milliporc Corp. or equivalent. 

jj Sigma Chemical Co., biological grade, or equivalent. 

# Cargillc Laboratories, Inc., Type B. or equivalent.